Microfluidic system with fluid pickups

ABSTRACT

Microfluidic system, including methods and apparatus, for processing fluid, such as by droplet generation. In an exemplary method, a sample-containing fluid may be dispensed into a well through a sample port of a channel component. The channel component may include (a) a body having a bottom surface attached to the well, and a top surface with a microchannel formed therein, and (b) an input tube projecting into the well from the bottom surface of the body. The sample-containing fluid when dispensed may contact a bottom end of the input tube and may be retained, with assistance from gravity, out of contact with the microchannel. A pressure differential may be created that drives at least a portion of the sample-containing fluid from the well via the input tube and through the microchannel.

CROSS-REFERENCES TO PRIORITY APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/230,757, filed Aug. 8, 2016, issued Jun. 27, 2017, as U.S. Pat. No.9,687,848. The '757 application, in turn, is a divisional of U.S. patentapplication Ser. No. 14/312,488, filed Jun. 23, 2014, issued Aug. 9,2016, as U.S. Pat. No. 9,409,174. The '488 application, in turn, isbased upon and claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application Ser. No. 61/838,063, filed Jun. 21, 2013.Each of these priority applications is incorporated herein by referencein its entirety for all purposes.

CROSS-REFERENCES TO OTHER MATERIALS

This application incorporates by reference in their entireties for allpurposes the following materials: U.S. Pat. No. 7,041,481, issued May 9,2006; U.S. Patent Application Publication No. 2010/0173394 A1, publishedJul. 8, 2010; U.S. Patent Application Publication No. 2011/0217711 A1,published Sep. 8, 2011; U.S. Patent Application Publication No.2012/0152369 A1, published Jun. 21, 2012; U.S. Patent ApplicationPublication No. 2012/0190032 A1, published Jul. 26, 2012; U.S. PatentApplication Publication No. 2014/0024023 A1, published Jan. 23, 2014;and Joseph R. Lakowicz, PRINCIPLES OF FLUORESCENCE SPECTROSCOPY (2^(nd)Ed. 1999).

INTRODUCTION

Many biomedical applications rely on high-throughput assays of samplescombined with reagents. For example, in research and clinicalapplications, high-throughput genetic tests using target-specificreagents can provide high-quality information about samples for drugdiscovery, biomarker discovery, and clinical diagnostics, among others.As another example, infectious disease detection often requiresscreening a sample for multiple genetic targets to generatehigh-confidence results.

The trend is toward reduced volumes and detection of more targets.However, creating and mixing smaller volumes can require more complexinstrumentation, which increases cost. Accordingly, improved technologyis needed to permit testing greater numbers of samples and combinationsof samples and reagents, at a higher speed, a lower cost, and/or withreduced instrument complexity.

Emulsions hold substantial promise for revolutionizing high-throughputassays. Emulsification techniques can create millions of aqueousdroplets that function as independent reaction chambers for biochemicalreactions. For example, an aqueous sample (e.g., 200 microliters) can bepartitioned into droplets (e.g., four million droplets of 50 picoliterseach) to allow individual sub-components (e.g., cells, nucleic acids,proteins) to be manipulated, processed, and studied discretely in amassively high-throughput manner.

Splitting a sample into droplets offers numerous advantages. Smallreaction volumes (e.g., picoliters to nanoliters) can be utilized,allowing earlier detection by increasing reaction rates and forming moreconcentrated products. Also, a much greater number of independentmeasurements (e.g., thousands to millions) can be made on the sample,when compared to conventional bulk volume reactions performed on amicroliter scale. Thus, the sample can be analyzed more accurately(i.e., more repetitions of the same test) and in greater depth (i.e., agreater number of different tests). In addition, small reaction volumesuse less reagent, thereby lowering the cost per test of consumables.Furthermore, microfluidic technology can provide control over processesused for the generation, mixing, incubation, splitting, sorting, anddetection of droplets, to attain repeatable droplet-based measurements.

Aqueous droplets can be suspended in oil to create a water-in-oilemulsion (W/O). The emulsion can be stabilized with a surfactant toreduce or prevent coalescence of droplets during heating, cooling, andtransport, thereby enabling thermal cycling to be performed.Accordingly, emulsions have been used to perform single-copyamplification of nucleic acid target sequences in droplets using thepolymerase chain reaction (PCR).

Compartmentalization of single copies of a nucleic acid target indroplets of an emulsion alleviates problems encountered in amplificationof larger sample volumes. In particular, droplets can promote moreefficient and uniform amplification of targets from samples containingcomplex heterogeneous nucleic acid populations, because samplecomplexity in each droplet is reduced. The impact of factors that leadto biasing in bulk amplification, such as amplification efficiency, G+Ccontent, and amplicon annealing, can be minimized by dropletcompartmentalization. Unbiased amplification can be critical indetection of rare species, such as pathogens or cancer cells, thepresence of which could be masked by a high concentration of backgroundspecies in complex clinical samples.

Despite their allure, emulsion-based assays present technical challengesfor high-throughput testing, which can require creation of tens,hundreds, thousands, or even millions of individual samples andsample/reagent combinations. Droplet generation, in particular, poses aspecial challenge. Current droplet generators require mechanisms forholding sample back from the point of droplet generation until a properpressure environment is established and oil is introduced to the dropletgeneration point. In the past, this has been done primarily byintroducing air traps in the sample line, which can require severalminutes of delay in wicking of the aqueous sample. It also has been doneby introducing valves into the microfluidic lines, which complicatesboth fabrication and ease of operation. Moreover, current dropletgenerators bubble air through the generated droplets after sampleprocessing has been completed, a phenomenon that is known to damagedroplets. Thus, there is a need for improved approaches for thegeneration of droplets.

SUMMARY

The present disclosure provides a microfluidic system, including methodsand apparatus, for processing fluid, such as by droplet generation. Insome embodiments, the system may include a well and a channel componentattached to the well. The channel component may include (a) a body, (b)an input tube (a “fluid pickup”) projecting from a bottom surface of thebody and having an open bottom end disposed in the input well, (c) amicrochannel, and (d) a passage extending through the input tube and thebody and connecting the well to the microchannel. The system may beconfigured to receive a sample-containing fluid in the well and retainthe sample-containing fluid below a top end of the passage, until apressure differential is created that drives at least a portion of thesample-containing fluid from the well via the passage and through themicrochannel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic sectional view of selected aspects of an exemplarymicrofluidic system for fluid processing and including a microfluidicdevice having an input tube projecting into an input well, with theinput tube in contact with a fluid sample contained by the input welland in communication with a microchannel disposed above the input well,and with the fluid sample retained in the well with the assistance ofgravity, in accordance with aspects of the present disclosure.

FIG. 2 is another schematic sectional view of selected aspects of themicrofluidic system of FIG. 1, taken after a pressure differential hasbeen created by application of a vacuum to the microfluidic device, todrive the fluid sample from the input well to an output well via theinput tube and the microchannel, in accordance with aspects of thepresent disclosure.

FIG. 2A is a schematic sectional view of selected aspects of a differentversion of the microfluidic system of FIG. 1, taken as in FIG. 1 andhaving one or more microchannels formed in a bottom surface of a cap ofthe channel component, in accordance with aspects of the presentdisclosure.

FIG. 2B is a schematic sectional view of selected aspects of anotherversion of the microfluidic system of FIG. 1, taken as in FIG. 1 andhaving a channel component with a two-layer cap, in accordance withaspects of the present disclosure.

FIG. 3 is a partially exploded view of an exemplary embodiment of themicrofluidic device of FIG. 1 constructed as a droplet generation devicehaving an array of emulsion production units and including a channelcomponent attached to and overlying a well component, with the channelcomponent including a base covered by a cap, and with the cap explodedfrom the base, in accordance with aspects of the present disclosure.

FIG. 4 is an exploded sectional view of the device of FIG. 3, takengenerally along lines 4-4 of FIG. 3 in the absence of the cap andshowing a single emulsion production unit of the device.

FIG. 5 is a fragmentary plan view of the base of the channel componentof the device of FIG. 3, taken around the single emulsion productionunit of FIG. 4.

FIG. 6 is a fragmentary bottom view of the base of the channel componentof the device of FIG. 3, taken as FIG. 5 except from the oppositedirection.

FIG. 7 is a fragmentary sectional view of the base and the wellcomponent of the device of FIG. 3, taken generally along line 7-7 ofFIG. 5 through a single emulsion production unit.

FIG. 8 is another fragmentary sectional view of the base and the wellcomponent of the device of FIG. 3, taken generally along line 8-8 ofFIG. 5 through a single emulsion production unit.

FIG. 9, presented as FIGS. 9A and 9B on separate pages, is a flowchartillustrating exemplary steps that may be performed in a method ofgenerating droplets with the device of FIG. 3, with fragmentary portionsof the device shown in cross section before and after performance ofeach step, in accordance with aspects of the present disclosure.

FIG. 10 is an isometric view of another exemplary embodiment of themicrofluidic device of FIG. 1 constructed as a droplet generation devicehaving an array of emulsion production units and including a channelcomponent attached to and overlying a well component, in accordance withaspects of the present disclosure.

FIG. 11 is an exploded isometric view of the microfluidic device of FIG.10.

FIG. 12 is a plan view of the well component of the device of FIG. 10.

FIG. 13 is a plan view of a base of the channel component of the deviceof FIG. 10, taken in the absence of a cap of the channel component.

FIG. 14 is a fragmentary plan view of a single emulsion production unitof the device of FIG. 10, taken generally around the region indicated at“14” in FIG. 13 in the absence of the cap.

FIG. 15 is a fragmentary sectional view of the channel component of thedevice of FIG. 10, taken generally along line 15-15 of FIG. 14 (but withmirror-image symmetry) through a carrier manifold and a pair of carrierchannels that extend from the carrier manifold to a pair of emulsionproduction units.

FIG. 16 is a fragmentary sectional view of the channel component of thedevice of FIG. 10 connected to a source of carrier fluid, takengenerally along line 16-16 of FIG. 14 through a carrier port of thedevice and illustrating a path for flow of carrier fluid into a channelnetwork of the device without passing through the bottom side of thechannel component, in accordance with aspects of the present disclosure.

FIG. 17 is a sectional view of a single emulsion production unit of thedevice of FIG. 10, taken generally along line 17-17 of FIG. 14 in thepresence of the cap.

FIG. 18 is another sectional view of the single emulsion production unitof the device of FIG. 10, taken generally along line 18-18 of FIG. 14 inthe presence of the cap.

FIG. 19 is a schematic view of an exemplary instrument for driving fluidflow for droplet generation within the device of FIG. 10, taken with theinstrument holding the device of FIG. 10 but not yet fluidicallyconnected to the device, and with the device viewed at elevation, inaccordance with aspects of the present disclosure.

FIG. 20 is a bottom view of a head of the instrument of FIG. 19, takengenerally along line 20-20 of FIG. 19.

FIG. 21 is a bottom view of another exemplary head for the instrument ofFIG. 19, taken as in FIG. 20.

DETAILED DESCRIPTION

The present disclosure provides a microfluidic system, including methodsand apparatus, for processing fluid, such as by droplet generation. Insome embodiments, the system may include a well and a channel componentattached to the well. The channel component may include (a) a body, (b)an input tube (a “fluid pickup”) projecting from a bottom surface of thebody and having an open bottom end disposed in the input well, (c) amicrochannel, and (d) a passage extending through the input tube and thebody and connecting the well to the microchannel. The system may beconfigured to receive a sample-containing fluid in the well and retainthe sample-containing fluid below a top end of the passage, until apressure differential is created that drives at least a portion of thesample-containing fluid from the well via the passage and through themicrochannel.

The present disclosure provides systems, including methods andapparatus, for generating droplets suitable for droplet-based assays.The systems may include (A) a channel component (e.g., a dropletgeneration component) for forming droplets, (B) a well component forholding sample before droplet generation and for holding generatedsample-containing droplets after droplet generation, and (C) an inputtube (and, optionally, an output tube) operatively disposed between thechannel component and the well component, for introducing sample intothe channel component before droplet generation (and, optionally, fordepositing generated droplets back into a respective well(s) of the wellcomponent after droplet generation, in optional anticipation of furtherprocessing and/or analysis). The flow of fluid into, within, and out ofthe system may be controlled by creation of a pressure differential, forexample, by application of a suitable vacuum.

The system may comprise a single-piece device (i.e., the device may beintegral or monolithic) or a device formed by two or more pieces (e.g.,the device may be modular). In the latter case, components of the devicemay be joined via any suitable mechanism, such as snapping or adhering,to form the complete device. The channel component may be positionedabove and/or over the well component in use, with pickup tubes disposedin between. A sample-containing fluid (interchangeably termed a“sample,” a “sample fluid,” or a “fluid sample”), a carrier fluid, andan emulsion may then be held in respective wells at least partially bygravity. In addition, sample-containing fluid (and, optionally, carrierfluid) may then be drawn up against gravity from a sample well (and,optionally, a carrier fluid well) through one or more input tube(s),processed to form droplets in the channel component, and then depositedback down into an emulsion well(s) through output tubes, in thedirection of, and with the possible assistance of, gravity. The systemmay be wholly or partially disposable (particularly portions of thesystem that contact sample and that would thus subject subsequentsamples to contamination).

The channel component configured as a droplet generation component mayinclude any suitable mechanism(s) for forming droplets. Typically, thiswill involve forming sample-containing droplets by merging an aqueous,sample-containing fluid with a carrier fluid, such as oil, to form anemulsion of sample-containing aqueous droplets suspended in the carrierfluid. Channels may be provided to transport the sample-containing fluidand carrier fluid and the sample-containing droplets within the dropletgeneration component. In some embodiments, the droplet generationcomponent may be at least substantially planar, with droplets generatedwithin a substantially planar channel network disposed in a dropletgeneration “chip component.”

The well component may comprise any suitable collection of reservoirsfor holding the respective fluids and droplets. Wells for sample andemulsion, and, optionally, carrier fluid and/or vacuum access, mayindependently be of any suitable size and shape. Wells in a givenembodiment may be of the same or different sizes and/or shapes, relativeto one another. For example, among other possibilities, wells forcarrier fluid and emulsion may be largest and deepest, the well forsample may be smallest and shallowest, and the well for vacuum accessmay be of intermediate size and depth.

The input and output (or pickup and deposit (or delivery (or deposit))tubes may comprise any suitable fluidic connection between the dropletgeneration component and the well component consistent with the pickupand deposit functions described above. In particular, they may formhollow fluidic connections between channels in the droplet generationcomponent and fluid wells in the well component, acting as fluid pickupsor “sippers” to obtain fluid from certain wells and/or to deliver fluidto other wells through the channels. The tubes, like the wells, mayindependently be of any suitable size (length and internal and externaldiameter) and shape. Moreover, in a given embodiment, the tubes may bethe same or different sizes and shapes, relative to one another. Thesample tube and, optionally, carrier fluid tube may be configured (e.g.,sized lengthwise) to maintain continuous contact with sample and carrierfluid, respectively, while the system is in use. The droplet or emulsiontube, in contrast, may be configured to avoid contact with droplets oncethey are deposited in the emulsion well (e.g., by allowing emulsion toseparate from the droplet tube and drip or fall into the emulsion well).Thus, if the carrier fluid and emulsion wells are of the same orcomparable size, the carrier fluid tube may be longer, and the emulsionwell tube may be shorter, relative to one another. The tubes may haveinternal diameters sufficient to reduce or prevent capillary movement ofsample and carrier fluid to the channel network of the dropletgeneration component. These internal diameters may vary, depending onthe fluid and on the composition of the (fluid-contacting portion ofthe) tube. The various tubes and the droplet generation component may beformed as one piece or from separate pieces that are joined together.

The system may be preconfigured in different formats, depending onintended use, intended user, and so on. For example, the system may bepre-loaded with carrier fluid, so that the user only needs to add sampleand a vacuum (or pressure) source. The system may include a pierceablecover, which may be pierced just prior to or simultaneous with theintroduction of sample, to reduce contamination. The system also may bereplicated, for example, on a microplate footprint, to allowsimultaneous processing of multiple samples and/or multiple replicatesof a single sample.

The system may provide significant advantages over current systems. Forexample, the input tube for a sample may be configured to allow gravityto limit the filling of the channel component with the sample,overcoming capillary action, and reducing or avoiding the need for airtraps and/or valves by holding the sample away from the channelcomponent. Alternatively, or in addition, the output tube(s), foroutputting processed fluid (e.g., an emulsion) from the channelcomponent may allow the processed fluid to drip into a receiving well,reducing or preventing bubbling of air through processed fluid.Additional features of the system may ease workflow. First, openchannels in the channel component may be fed through a central locationso that they can be resealed after fluid processing (e.g., dropletgeneration) through a melting process, termed “heat staking.” Second,wells of the well component may be fluidically connected, for example,via a microchannel, to enable venting of both wells with a singlepuncture of a penetrable cover of the channel component. Third, thevacuum tube, or dripper, if present, may reduce the likelihood that thevacuum interface will be contaminated, even if there is an overflow.

Droplet generation systems according to the present disclosure may bepart of an overall assay system configured to test for the presence of atarget (e.g., a target molecule or target sequence) in a sample. Theseoverall systems may include methods and apparatus for (A) preparing asample, such as a clinical or environmental sample, for analysis, (B)separating copies of the target by partitioning the sample into dropletseach containing no copies or one or more copies of the target (such as asingle copy of a nucleic acid target or other analyte of interest), (C)amplifying or otherwise reacting the target within the droplets, (D)detecting the amplified or reacted target, or characteristics thereof,and/or (E) analyzing the resulting data. In this way, complex samplesmay be converted into a plurality of simpler, more easily analyzedsamples, with concomitant reductions in background and assay times.Exemplary systems (including exemplary droplet generators) are describedin the patent documents listed above under Cross-References andincorporated herein by reference.

Additional features of fluid processing systems according to the presentdisclosure, as well as exemplary embodiments, are described in thefollowing sections: (I) overview of microfluidic systems for fluidprocessing, and (II) examples.

I. OVERVIEW OF MICROFLUIDIC SYSTEMS FOR FLUID PROCESSING

This section provides an overview of microfluidic systems for processingfluid and including an input well and a microchannel in fluidcommunication with the input well via an input tube extending into theinput well; see FIGS. 1 and 2.

FIG. 1 shows selected aspects of an exemplary microfluidic system 50 forfluid processing. The system may include a microfluidic device 52 tohold fluid and direct fluid flow, a fluid-transfer device 54 tointroduce fluid into and/or remove fluid from device 52, and at leastone vacuum/pressure source 56, which may include at least one pump, todrive fluid flow within the device.

Device 52 may include a fluid-holding portion 58 that is attached orattachable to a fluid-processing portion 60. The fluid-processingportion, and particularly a microfluidic region thereof, may be disposedabove, and optionally over, the fluid-processing portion.

Fluid-holding portion 58 may include one or more reservoirs 62 to holdone or more fluids such as at least one sample-containing fluid 64, acarrier fluid (interchangeably termed a carrier), an emulsion includingsample-containing droplets (interchangeably termed sample droplets)disposed in the carrier, or any combination thereof, among others. Eachfluid may include or be liquid and may be held in the fluid-holdingportion for a variable/desirable time before and/or after the fluid isintroduced into and/or received from the fluid-processing portion.

The fluid-holding portion may include at least one well component 66that provides at least one well. For example, well component 66 mayprovide at least one input well 68 to hold fluid for introduction intofluid-processing portion 60 and at least one output well 70 to collectand hold fluid received from one or more input wells via thefluid-processing portion. In some embodiments, the input well and theoutput well may be formed by discrete well components, and the outputwell may not be attached to the fluid-processing portion and/or may beremovable from the fluid-processing portion. For example, the outputwell may be provided by a receptacle, such as a tube, that is removablefrom the fluid-processing portion for further processing, such asthermocycling to promote nucleic acid amplification. In someembodiments, well component 66 may be molded, such as injection molded,as a single piece. The well component may be formed of polymer, such asplastic.

Fluid-processing portion 60 may include a channel component 72 thatincludes one or more channels 74. In some embodiments, the channelcomponent may be described as a droplet generation component. Eachchannel 74 may be enclosed, at least between its ends, by channelcomponent 72. At least one channel 74 may be a “microchannel” (alsotermed a “microfluidic channel”), namely, any channel having acharacteristic transverse dimension (e.g., a diameter) of less than onemillimeter. A “microfluidic device” and a “microfluidic system” eachhave at least one microchannel.

Each channel, whether or not microfluidic, can be circumferentiallybounded between its ends (i.e., a bounded channel), or may be open onone side (e.g., a groove that is bounded below and on both lateral sidesbut open above (i.e., an unbounded or uncapped channel)). The samechannel may be described as having a conceptually (or literally)unbounded form (e.g., a groove formed in a base (or cap) of the channelcomponent) and a bounded form (e.g., the groove covered by a cap (orbase) attached to the base (or cap)).

In some embodiments, the channel component may include a plurality offluidically connected channels forming a channel network. The channelnetwork may provide a channel intersection at which two or more channelsmeet to provide a droplet generator (interchangeably termed a site ofdroplet generation). The channel network and/or one or more channels 74may be planar. The channel network and/or one or more channels 74, suchas one or more microchannels, may be horizontal.

Channel component 72 may be formed integrally as a single piece, or maybe composed of two or more pieces that are formed separately and thenattached to one another. For example, channel component 72 may include alower member 76 (interchangeably termed a base) and an upper member 78(interchangeably termed a cap or cover). Base 76 may include a body 80and one or more tubes, such as at least one input tube 82 and/or atleast one output tube 84. In some embodiments, base 76 may be molded,such as injection molded, as a single piece. The base may be formed ofpolymer, such as plastic.

Each tube may be attached to body 80. The tube may be formed integrallywith the body, or formed separately and then attached to the body. Ineither case, the tube may be permanently attached to the body and fixedin position with respect to the body. An input tube interchangeably maybe described as a straw, a sipper, or a pickup. An output tubeinterchangeably may be described as a dripper.

The body may have any suitable structure. The body may be described as asubstrate, a chip, or a chip component. The body may be planar. A bottomsurface 86 of the body may be attached to well component 66, such as toform a fluid-tight seal between the body and one or more wells (and/oreach well) of the well component. The body and a well collectively mayform a chamber to hold fluid, with body 80 and/or channel component 72forming a ceiling of the chamber and/or a cover for the well. Body 80may at least partially bound each channel 74. A top surface 88 of thebody may have at least a lower portion of each channel 74 formedtherein. For example, a bottom wall and opposing lateral side walls (orlower side wall regions) of channel 74 may be formed in top surface 88.In some embodiments, a bottom surface of the cap alternatively or alsomay have one or more channels formed therein (see below). The topsurface and the bottom surface each may be substantially planar.

Body 80 and input tube 82 collectively may form a passage 90 thatfluidically connects the inside of input well 68 to a channel 74. Thepassage may extend upward from an open bottom end 92 of input tube 82 tothe channel, and may be considered as being formed by a hole throughinput tube 82 that joins an aperture through body 80. Accordingly, thepassage may extend through base 76 from a bottom side to a top side ofthe base. The passage may (or may not) be vertical or at least generallyvertical and may be arranged transverse (e.g., substantially orthogonal)to at least one channel 74 and/or a channel network formed by channels74. Bottom end 92 of input tube 82 may be positioned near the bottom ofinput well 68, to maximize the uptake of fluid from the input well.

Body 80 and output tube 84 also may define a passage 94 that fluidicallyconnects output well 70 to at least one channel 74. However, in someembodiments, the output tube may extend only into the upper region ofthe output well (or may be omitted altogether).

Channel component 72 also may define one or more ports, such as an inputport 96 and an output port 98. Input port 96 may be formed over theinput well and may be contiguous therewith, and provides access to theinterior of input well 68 for introduction of sample 64. Accordingly,the input port may be dimensioned to receive a bottom end portion offluid-transfer device 54, before or as the fluid-transfer devicedispenses the sample into the input well. Output port 98 may bestructured like the input port and provides access to the interior ofoutput well 70 to allow removal of fluid with another fluid-transferdevice 54. The ports shown here are through-holes that extend throughbody 80 between its top and bottom surfaces. In some embodiments, one ormore other ports may be constructed as blind holes in the body that arecontiguous with a channel formed in a top surface of the body (e.g., seeExample 2).

Cap 78 may be attached to top surface 88 of body 80 in a fluid-tightseal. The cap may be termed a cover, a sealing member, or a cappingmember, and may be substantially planar. The cap may at least partiallybound each channel 74. The cap may form a top wall of each channel, and,in some embodiments, side walls or at least side wall regions of eachchannel. The cap may be a cover formed as only one layer, or two or moreoverlapping (or nonoverlapping) layers of material. If the cover iscomposed of two or more layers, the layers may be connected to the bodyat the same time or sequentially. For example, the cover may include afirst layer attached directly (e.g., bonded) to a top surface of thebody and a second layer applied later (e.g., to cover one or moreopenings defined by the first layer). The cover may (or may not) besubstantially thinner than the body, such as less than 20%, 10%, or 5%of the body thickness. In some embodiments, the cover may be thickerthan the body.

The cap may cover any suitable openings formed in the top surface of thebody. For example, the cap may cover the top end of each passage 90, 94,and may or may not cover each port 96, 98. In some embodiments, the capmay be configured to be pierced at one or more ports during use of thesystem. Accordingly, any of the ports may be provided as a closed portthat can be opened by the user (and/or an instrument) by piercing thecap, which may performed to gain access to an underlying well, and/or tocreate fluid communication with one or more channels 74. The port may beopened to form a vent and/or a fluid transfer point.

Wells or well protrusions for holding fluid may be formed as one or morewell components 66 which are initially separate from body 80 of channelcomponent 72 and which are configured to form a substantially fluidtight seal or interface with the body. In other cases, some or all ofthe wells may be integrally formed with the body. The well component andthe body (and/or channel component) may be configured to mate together.In some cases, the well component and the body (and/or channelcomponent) may be sealed together by a user or during manufacture, toform a substantially fluid tight connection. Furthermore, as describedin more detail below, one or both of the well component and the body(and/or channel component) may have a larger format or “footprint” thanis depicted in FIG. 1, such as a microplate format, to allow two or more(or many) different samples to be processed (e.g., used to generatedroplets) in one system. Components of the system may work together to(A) pick up sample from a sample well, (B) introduce carrier fluid froma carrier reservoir, (C) bring these components together to generatedroplets, and (D) dispense the droplets to an emulsion well.

Fluid-transfer device 54 may be any device or set of devices capable ofdispensing and/or picking up fluid. Device 54 thus may be a pipette, asyringe, or the like. The device may be operated manually orautomatically.

Pressure source 56 may be any device or set of devices capable ofcreating a pressure differential within device 52. The pressure source56 may apply (positive) pressure to the device to push fluid, and/or avacuum (also called suction) to the device to pull fluid. The pressuresource may be capable of forming a sealed connection or sealedengagement with device 52, such as via a gasket 100 and/or at least onepiercing element 102 that pierces cap 78, among others. The system alsoor alternatively may include one or more other piercing elements 102that are operatively positioned or positionable over other ports, suchas to vent the system, access or fluidically connect to the ports, orthe like. The piercing element may be hollow (e.g., a pointed tube) topermit fluid flow through the pierced cap while the piercing elementremains in place.

FIG. 1 shows input tube 82 of base 76 extending into an input well 68that contains a volume of sample fluid 64. This arrangement allowsconfinement of the sample fluid by gravity. When a tube is in contactwith a fluid (e.g., sample fluid 64) in a reservoir (e.g., well 68),gravity may limit the ability of the fluid to travel up the tube. Thecapillary rise height H depends on the tube inner radius r, the surfacetension y of the fluid at a liquid-air interface, the density of thefluid p, and the contact angle θ of the fluid on the tube surface,according to the following formula:

$H = \frac{2\gamma\mspace{11mu}\cos\mspace{11mu}\theta}{\rho\;{gr}}$

Provided that the capillary rise height is less than the heightdifference between the fluid in the well and a channel 74 above thewell, gravity will ensure that the fluid cannot reach the channelwithout application of a driving force, such as a pressure differential.Accordingly, the fluid is retained in the well until the driving forceis applied.

In a more specific example, intended only for illustration, assume thatchannel 74 is located 6 mm above the bottom of the well, and the heightof the fluid in the well is 3 mm. The capillary rise for an illustrativesample for PCR may be calculated as approximately 1.3 mm for a 1 mminternal diameter tube. This arrangement positions the sample meniscusinside the tube at 1.7 mm below channel 74.

In some cases, the volume (e.g., the well) into which the tube extendsmay be closed instead of vented, increasing the retention power of thestructures described above. Any capillary rise would be counteracted byan increase of back-pressure in the closed volume, further isolating thefluid in the well from channel 74 above.

FIG. 2 shows system 50 taken after a pressure differential has beencreated by application of a vacuum to device 52 with pressure source 56.The pressure differential may create pneumatic flow into device 52 (suchas flow of air into the input well), indicated by an arrow at 104, andout of device 52 (such as flow of air out of the output well), indicatedby an arrow at 106. A pressure difference causes sample fluid 64 totravel up input passage 90, against the force of gravity, indicated byan arrow at 108, from the bottom end of input tube 82. The sample fluidexits input passage 90 and flows through at least one channel 74,indicated by an arrow at 110, and then travels downward, in thedirection of gravity, indicated by arrow at 112, through output passage94 and into output well 70. Outputted fluid 114 is collected in theoutput well. In the depicted embodiment, air enters device 52 at aposition over input well 68 and exits the device at a position overoutput well 70. In other embodiments, air may enter and/or exit thedevice at a position that is above but not over the corresponding well,via a horizontally offset vent port or vacuum port.

In some embodiments, the pressure differential drives a carrier fluid116, indicated by an arrow at 118, from a source of carrier fluid. Thesource may be within device 52 or may be external to the device (e.g.,originating from carrier reservoir 120). The source of carrier fluid maybe fluidically connected to one or more channels 74 such that samplefluid 64 and carrier fluid 116 are driven through a channel intersection122 formed where a plurality of channels 74 meet one another.Sample-containing droplets 124 disposed in carrier fluid 116 (acontinuous phase) may be formed at channel intersection 122 andcollected in output well 70 (e.g., see Examples 1 and 2).

The channel component may define an open path for fluid flow from theinput well to the output well. The path may remain open (unobstructed)as the input well is loaded with sample, while the input well retainsthe loaded sample with the assistance of gravity, and as the sample isdriven to the output well. Accordingly, the channel component and/or theflow path may be described as being valve-less and/or as havingvalve-less microfluidics.

FIG. 2A shows a different version of microfluidic system 50 of FIG. 1.The system of FIG. 2A is similar to that of FIG. 1, except that one ormore channels 74, such as at least one microchannel and/or a channelnetwork for droplet generation, are formed at least partially in abottom surface 126 of cap 78 of channel component 72. The bottom surfacemay be substantially planar. Each channel 74 of device 52 of the systemmay be formed in bottom surface 126 of cap 78 instead of in a topsurface 88 of base 76. The top surface also may be substantially planar.Base 76 may form a bottom wall of each channel 74.

FIG. 2B shows another version of microfluidic system 50 of FIG. 1. Thesystem of FIG. 2B is similar to that of FIG. 2A, except that cap 78 iscomposed of more than one layer, such as an upper layer 128 (e.g., anupper sheet of material) attached to a lower layer 130 (e.g., a lowersheet of material). Upper layer 128 may be similar or identical to cap78 of FIG. 1. The upper layer may be substantially planar and/or mayhave a substantially planar bottom surface forming a top wall of one ormore channels 74. Lower layer 130 may be substantially planar and may besandwiched between upper layer 128 and base 76. The lower layer may formlateral side walls of each channel 74 but neither the top wall nor thebottom wall of each channel. Base 76 may form a bottom wall of eachchannel 74.

Further aspects of exemplary droplet generation systems that may besuitable for the present fluid processing systems, includingmicrofluidic devices, droplet generators, samples, carrier fluids,droplets, emulsions, droplet-based assays, instruments to drive andcontrol droplet generation, and methods of droplet generation, amongothers, are described in the patent documents listed above underCross-References, which are incorporated herein by reference.

II. EXAMPLES

This section describes selected aspects and embodiments of the presentdisclosure related to systems and methods for fluid processing and/ordroplet generation. These examples are intended for illustration onlyand should not limit or define the entire scope of the presentdisclosure.

Example 1. Droplet Generation Device with Fluid Pickups for Sample andCarrier

This example describes an exemplary embodiment 152 of microfluidicdevice 52 for fluid processing system 50 (see FIGS. 1, 2, 2A, and 2B)that is configured for generating an array of emulsions, and alsodescribes exemplary methods of using device 152 to generate and processemulsions; see FIGS. 3-9. The emulsions are generated with samples andcarrier fluid transported from wells to channels via fixed, dedicatedpickup tubes.

Device 152 uses hollow protrusions or “fluid pickups” in conjunctionwith droplet generators, and provides specific examples of dropletgenerators in which sample-containing droplets suspended in a carrierfluid are generated and transported substantially within a plane.

As used herein, “substantially within a plane” or “substantially planar”with respect to droplet generation means that the radius of curvature ofthe space in which droplets are generated and transported is muchgreater than the cross-sectional dimensions of the channels throughwhich the droplets are created and transported, and the curvature doesnot substantially alter the hydraulic function of the channels.

FIG. 3 shows a partially exploded view of device 152. The device has awell component 66 attached to and underlying a channel component 72. Thechannel component has a base 76 that is attached to wells of wellcomponent 66 to form a fluid-tight circumferential seal at the topperimeter of each of the wells. A cap 78 of channel component 72 isshown exploded from base 76, although the cap is typically pre-attachedto a top surface 88 of base 76 before use, such as during manufacture ofdevice 152.

Device 152 forms an array of emulsion production units 154. The depictedembodiment has a two-by-four array of units 154, although the device mayhave any suitable number of the units. The emulsion production units maygenerate emulsions in parallel, in this case, a set of eight emulsionsin parallel. The emulsion production units may be replicates of oneanother and may be arranged as an SBS-compatible array, such as with aunit repeated every 18, 9, 4.5, 2.25, or 1.125 mm, among others, alongeach row and column of units 154.

FIG. 4 shows one of emulsion production units 154 in the absence of cap78. Channel component 72 includes a channel network 156 of channels 74formed in top surface 88 of a planar body 80 and capped with cap 78 suchthat each channel is circumferentially bounded along its length. Aplurality of tubes project from a bottom surface 86 of the body, namelya sample tube 158, a carrier tube 160, a droplet tube 162, and a vacuumtube 164. Tubes 158 and 160 are input tubes 82, tube 162 is an outputtube 84 (see FIGS. 1 and 2), and tube 164 is an optional output-liketube to further separate the vacuum interface from the emulsion. Each oftubes 158, 160, 162, and 164 extends into a respective well of wellcomponent 66: tube 158 into a sample well 166 (an input well 68), tube160 into a carrier well 168 (another input well 68), tube 162 into anemulsion well 170 (an output well 70), and tube 164 into a vacuum well172. In other embodiments, the channels may be formed in a bottomsurface of cap 78 (e.g., see FIGS. 2A and 2B).

FIG. 5 shows channel network 156 in plan view. The network includes asample channel 174, a plurality of carrier channels 176 and 178, and adroplet channel 180, which intersect at a channel junction 182(interchangeably termed a droplet generation site) where droplets aregenerated. (The channels are too small to be visible in the sectionalviews of FIGS. 6-9.) Sample channel 174 carries sample fluid to junction182, carrier channels 176 and 178 carry carrier fluid to the junction,and droplet channel 180 carries droplets of the sample fluid in carrierfluid from the junction. In other embodiments, only one carrier channelis present per unit 154.

Base 76 defines a plurality of apertures each extending through the basefrom a top side to a bottom side of the base (see FIGS. 5-8). Eachaperture may extend only through body 80 or through the body and one oftubes 158, 160, 162, or 164.

Ports may be created by apertures that extend only through the body: asample port 184 (an input port 96), a carrier port 186 (another inputport 96), an emulsion port 188 (an output port 98), and a vacuum port190. A vent channel 192 formed in a top surface of body 80 may connectthe sample port and the carrier port, to allow either port to vent theother port. Another channel 194 formed similarly helps to fluidicallyconnect emulsion port 188 with vacuum port 190 via vacuum passage 202.

Sample port 184 is configured to receive a sample, typically in fluidform. For example, sample-containing fluid may be inserted into sampleport 184 with a pipette, either manually or as part of an automatedsystem. Sample well 166 of well component 66 is disposed directly undersample port 184, as depicted in FIGS. 6 and 7, so that fluid insertedinto the sample port will pass into the sample well.

Carrier port 186 is configured similarly to receive oil or some othercarrier fluid. For example, as in the case of the sample-containingfluid, carrier fluid may be inserted into carrier port 186 with apipette, either manually or as part of an automated system. Carrier well168 is disposed directly under carrier port 186, as depicted in FIGS. 6and 7, so that carrier fluid inserted into the carrier port will passinto the carrier well.

Emulsion port 188 is configured to receive an emulsion extractor, suchas a needle, pipette, or syringe tip, which can be used to extract theemulsion resulting from the application of vacuum pressure at vacuumport 190. Emulsion well 170 is disposed directly under emulsion port188, and is configured to receive an emulsion of sample-containingdroplets in a manner to be described in more detail below.

Vacuum port 190 (interchangeably termed a vacuum interface) isconfigured to receive a vacuum connection, such as an end of a vacuumconduit, to apply negative pressure to the vacuum port. As will bedescribed in more detail below, the application of vacuum pressure atvacuum port 190 causes the formation of an emulsion of sample-containingdroplets suspended in carrier fluid, and transports the emulsion toemulsion well 170. Vacuum well 172 may be disposed directly under vacuumport 190. Passages may be created by apertures that extend through thetubes: a sample passage 196 (an input passage 90), a carrier passage 198(another input passage 90), a droplet passage 200 (an output passage94), and a vacuum passage 202.

Passages 196, 198, 200, and 202 are each in direct fluid communicationwith one of the channels of channel network 156, as depicted in FIG. 5.This configuration provides fluid and pressure communication betweenchannel network 156 and the wells of well component 66, as describedbelow.

Sample passage 196 extends through sample tube 158, which also may bereferred to as a “sample sipper.” When well component 66 and base 76(and/or channel component 72) are joined together, sample tube 158 isconfigured to fit within sample well 166, which allows the sample tubelater to pick up or “sip” sample-containing fluid from the sample well,and to transport the sample-containing fluid to the top of samplepassage 196 and thus to channel network 156. The internal diameter ofthe sample tube may be large enough to prevent significant capillarymovement of sample to a level much above that of the sample in the welloutside the tube. As shown in FIG. 7, sample tube 158 extends to aposition near the bottom of sample well 166, such that the bottom end ofsample passage 196 can pick up at least most of the sample in the well.

Carrier passage 198 extends through carrier tube 160, which also may bereferred to as a “carrier sipper.” When well component 66 and base 76(and/or channel component 72) are joined together carrier tube 160 fitswithin carrier well 168, which allows the carrier tube later to pick upor “sip” carrier fluid from the carrier well, and to transport thecarrier fluid to the top of carrier passage 198 and thus to channelnetwork 156. The internal diameter of the carrier tube may be largeenough to prevent significant capillary movement of carrier fluid to alevel much above that of the carrier fluid in the carrier well outsidethe tube. As shown in FIG. 7, carrier tube 160 extends to a positionnear the bottom of carrier well 168, such that the bottom end of carriertube 160 can pick up at least most of the carrier fluid in the well.

Droplet passage 200 extends through droplet tube 162, which may bereferred to as a “droplet dripper.” The droplet tube fits withinemulsion well 170 and is configured to transport an emulsion ofsample-containing droplets suspended in carrier fluid from channelnetwork 156 (and more particularly, droplet channel 180) and intoemulsion well 170, from which the emulsion may be extracted as describedpreviously. Transport of the droplets from the channel network into thedroplet dripper and then into the emulsion well results from negativepressure applied at vacuum port 190. As shown in FIG. 8, droplet tube162 does not extend to the lower portion of the emulsion well, to reducethe chance of drawing part of the emulsion to the vacuum well viaemulsion port 188, channel 194, and vacuum tube 164.

Vacuum passage 202 extends through vacuum tube 164, which also may bereferred to as a “vacuum dripper.” The vacuum tube fits within vacuumwell 172 and is configured to provide pressure communication between thevacuum well and the emulsion well via channel 194 and emulsion port 188.Vacuum pressure introduced into vacuum port 190 thus may be communicatedto sample sipper 158 and carrier sipper 160 through vacuum dripper 164and channel network 156, causing sample-containing fluid and carrierfluid to be drawn into the channel network through the sample sipper andthe carrier sipper, respectively.

A primary cap (such as cap 78) and/or a secondary cap (such as anotherlayer placed on the primary cap) may be placed on and/or over the topsurface of body 80, to seal the top of any suitable combination ofapertures defined by the body alone or in combination with one or moretubes. For example, the cap may seal channel network 156 or a portionthereof (such as any combination of sample channel 174, carrier channels176 and 178, and droplet channel 180), one more ports (such as sampleport 184, carrier port 186, emulsion port 188, and/or vacuum port 190),and one or more passages (such as sample passage 196, carrier passage198, droplet passage 200, and/or vacuum passage 202). Each cap may be asubstantially planar sheet, optionally treated with an adhesive on thebottom side and optionally having a substantially featureless bottomsurface. At least one cap may be applied during manufacture and/or atleast one cap may be configured to be applied by a user.

The cap may be pierced by a dedicated instrument or tool, a syringe,and/or a pipette tip, among others. Piercing the cap may create an airintake vent for a port, such as sample port 184 and/or carrier port 186,or an air outflow vent for emulsion port 188 (if pressure is used todrive fluid flow), among others. Alternatively, or in addition, piercingthe cap may create an access point for addition of sample to a samplewell or carrier fluid to a carrier well, or removal of emulsion from anemulsion well, among others.

In other cases, piercing the cap may create an opening over a vacuumport, such as vacuum port 190. For example, the aperture may be formedby pressing an end of a vacuum conduit through a cover (e.g., only aprimary cap, a primary cap and a secondary cap, or both, among others)and into the vacuum port, or by any other suitable method. In any case,the opening may provide an access point for a vacuum source to applyvacuum pressure to the device. As will be described in detail below,this causes formation of an emulsion of sample-containing dropletssuspended in carrier fluid, as well as transport of the emulsion throughsample sipper 158 and into emulsion well 170.

FIG. 9 shows a flowchart illustrating exemplary steps that may beperformed in a method of forming and processing an emulsion with device152. The device is depicted here in schematic cross section, to placetubes 158 and 160 and ports 184 and 186 in the same plane, and to placetubes 162 and 164 and ports 188 and 190 in the same plane. Sample port184 may be open on top, as shown, to provide access to sample well 166from above device 152, or cap 78 may be pierced to provide that access.

Sample 64 may be introduced into device 152, indicated by an arrow at220. Sample 64 may be dispensed to sample well 166 through sample port184, and then the port optionally may be covered with a cover element222. In exemplary embodiments, the cover element is a user-appliedadhesive film (e.g., an adhesive tape), which may cover any suitablenumber of openings in cap 78. The cover element ensures containment ofthe sample and reduces the chance of cross-contamination with differentsamples among the emulsion production units. As shown, sample 64 isadded to a level that is substantially below the top of the sample welland the top of sample passage 196. Gravity retains the sample in thisposition, allowing each of the sample wells of the device to be loadedwith sample before proceeding with droplet generation.

Carrier fluid 116 may be introduced into device 152, indicated by anarrow at 224. The carrier fluid may be dispensed into carrier well 168through carrier port 186, before or after the sample is dispensed intothe sample well. The device may be supplied to a user with carrier port186 open at the top (e.g., with an opening 226 defined in cap 78 overthe carrier port), or cap 78 may be pierced (manually by the user orautomatically with an instrument) to form an opening that opens thecarrier port for introduction of carrier fluid through the cap. Thecarrier fluid, similar to the sample, may be retained in the carrierwell by gravity, and below the top of carrier passage 198 inside thepassage, until driven out of the carrier well by a pressuredifferential. Alternatively, the device may be configured such that thecarrier fluid can flow to the channel network without a pressuredifferential. In other embodiments, the carrier fluid may be introducedmore directly into the channel network of the device through cap 78(e.g., from an external reservoir), without passing through base 76 (seeExample 2). In other words, carrier well 168 may be omitted from thedevice.

Vacuum may be applied, indicated by an arrow at 228. The vacuum createsa pressure differential that drives sample 64 and carrier fluid 116upward through respective passages 196, 198 into the channel network andthrough a channel intersection thereof to form droplets, and downthrough droplet passage 200 for collection as an emulsion 230 inemulsion well 170. Application of vacuum may be performed on emulsionproduction units 154 of device 152 in parallel to form a set of dropletswith each unit and collect an emulsion from each unit in parallel.

Each emulsion well may be sealed, indicated at 232. The emulsion wellmay, for example be sealed by blocking each channel that communicateswith the well by applying a heat stake 234 across a portion of thechannel network. Heat stake 234 seals (i.e., closes off (blocks)) thechannels across which it is disposed, for example, by melting orotherwise deforming the channel walls so that fluid can no longer passthrough the channels. Accordingly, heat stake 234 serves to fluidicallyisolate the emulsion well from the atmosphere and from the rest of thesystem, such as from the fluid contents of other wells and the rest ofthe channel network. Similarly, heat stake 234 fluidically isolates thevacuum port, the vacuum well, and the vacuum dripper from the remainingportions of the system. Furthermore, the heat stake partiallyfluidically isolates the sample port, the sample well, and the samplesipper. The only remaining fluid connection of these sample-relatedcomponents of the system to other portions of the system is through thechannel network to the carrier well.

Fluidic isolation of various components of the system from each otherthrough heat staking, as described above, or by any other means, may beperformed after the system is used to generate an emulsion ofsample-containing droplets, to insure that the emulsion will remain inthe emulsion well, and will not be drawn or otherwise transported backinto the channel network. Furthermore, sample-containing fluid will notbe able to pass inadvertently to the vacuum port, where it couldpotentially contaminate the vacuum hose or other fittings, potentiallyallowing those components to be reused rather than discarded after eachuse. Another purpose of the fluidic isolation may be to prevent loss offluid, such as carrier fluid or sample fluid (e.g., water) from theemulsion during thermal cycling, which may expose the emulsion totemperatures sufficient to cause evaporation.

Each emulsion may be thermally cycled (interchangeably termed“thermocycled”), indicated by an arrow at 236. After heat staking orsome other form of fluidically isolating the emulsion well, device 152may be placed in a thermocycler and thermocycled to cause theamplification of any target present in the emulsion within the emulsionwell. Finally, an opening may be formed in cap 78 above the emulsionwell, over the emulsion port, to open the emulsion port and provideaccess to the emulsion in the emulsion well. This allows an emulsionextraction tool, such as a pipette, needle, or syringe tip, to beinserted into the emulsion well and to extract the thermocycledemulsion.

After extraction from the emulsion well, the emulsion may be transportedto a detection system or region configured to detect the amplificationof a target in the droplets of the emulsion, for example, by detectingfluorescence radiation emitted by the droplets. In some cases, thesample-containing emulsion may be extracted from the emulsion well priorto thermocycling rather than after thermocycling, in which case theemulsion could be thermocycled while disposed within the extractioninstrument or after being placed in another suitable container.

The following description presents further exemplary methods ofoperating droplet generation systems. The methods may be generallysuitable for use with various droplet generation systems describedherein, at least including any of the systems shown in FIGS. 1-9 anddescribed in the accompanying text above and in Example 2 below. Thesteps presented below and elsewhere herein may be performed in anysuitable order and in any suitable combination (includingsubcombinations consisting of a subset of the steps). In someembodiments, steps may be repeated. Furthermore, the steps may becombined with and/or modified by any other suitable steps, aspects,and/features of the present disclosure. The following description refersto FIGS. 3-9 to indicate the state of an exemplary droplet generationsystem after various steps of the disclosed methods are performed, andto describe certain details of a suitable channel network that can beused in conjunction with the present methods.

Sample-containing fluid may be introduced through a sample port of achannel component (e.g., channel component 72) to a sample well (e.g.,sample well 166; see FIG. 9A, step 220). In some embodiments,sample-containing fluid may be added directly to the sample well, beforethe channel component (and/or a base thereof, such as base 76) is joinedwith the well component, thereby bypassing the sample port. Sample maybe added before or after attachment of a cap (e.g., cap 78) to a base ofthe channel component.

Carrier fluid may be transported through a carrier port of the channelcomponent to a carrier well (e.g., see step 224 of FIG. 9A). In someembodiments, carrier fluid may be added directly to the carrier well,before the channel component is joined with the well component, therebybypassing the carrier port. Carrier fluid may be added before or afterattachment of the cap.

A cap may be disposed or applied over the top surface of the base of thechannel component, to seal the top of apertures (e.g., ports andpassages) and a channel network formed in the top surface of the base.In some cases, the top of the channel network already may be sealed witha first cap, in which case a second cap may be applied to cover and/orseal one or more apertures, such as at least one sample port. The capmay be pierced to open a carrier port in the channel component and thusto allow the ingress of air into the carrier well. The cap may bepierced to open and/or fluidically connect to a vacuum port in thechannel component.

A vacuum source may be connected to the vacuum port. In some cases, theconnection may be achieved at least in part by piercing a cap over thevacuum port. In some cases, the end of a vacuum conduit may be used topierce the cap and access the vacuum port.

Negative pressure may be applied to the vacuum port by the vacuum source(e.g., see step 228 of FIG. 9B). This causes negative pressure in aninverted vacuum dripper of the channel component, such as vacuum tube164 depicted in FIG. 9B, and thus in a vacuum channel of the channelnetwork, such as vacuum channel 194 depicted in FIG. 5. Because the topof emulsion port 188 is sealed, this creates negative pressure in adroplet dripper of the chip component such as droplet tube 162 depictedin FIG. 9B, and thus in the channels of the droplet generator, as willnow be described in more detail with reference again to FIGS. 5-8.

Negative pressure in droplet tube 162 causes negative pressure in bothsample sipper 158 and carrier sipper 160 as follows. Exposed carrierport 186, which was opened in step 224 of FIG. 9A, allows the ingress ofair through the carrier port and into carrier well 168, which forcescarrier fluid through inverted carrier sipper 160 of the channelcomponent and into the channel network. Furthermore, sample vent channel192 provides fluid communication between the carrier port and the sampleport, which allows the ingress of air through the sample port and forcessample-containing fluid through the inverted sample sipper tube and intothe channel network.

More specifically, negative pressure in sample sipper 158 causessample-containing fluid to be drawn from the sample sipper into the topregion of sample passage 196, and from there into sample channel 174 ofthe channel network (see FIGS. 5 and 6). Similarly, negative pressure incarrier sipper 160 causes carrier fluid to be drawn from the carriersipper into a top region of carrier passage 198, and from there into apair of carrier fluid channels 176, 178 of the channel network. Thesample-containing fluid and the carrier fluid drawn into the channelnetwork meet at a cross-shaped droplet generation region, formed bychannel junction 182.

Droplets of sample-containing fluid suspended in carrier fluid aregenerated at the droplet generation region, in a manner describedpreviously. The cross-type droplet generation region shown in FIG. 5 ismerely exemplary. In some cases, other configurations of the same numberor a different number of intersecting channels may be used to generatedroplets, and all such configurations are within the scope of thepresent disclosure.

The emulsion of generated droplets may be transported through a dropletchannel 180 of the channel network to droplet passage 200, into dropletdripper 162, and into emulsion well 170.

One or more channels extending to the emulsion well may be deformed tofluidically isolate the emulsion well. For example, a heat stake may beapplied to the channel network of the channel component, to fluidicallyisolate the emulsion well (e.g., see step 232 of FIG. 9B). Equivalently,one could describe this step as “heat staking” an appropriate region ofthe channel network. For example, a heat stake may be applied acrossdroplet channel 180, sample vent channel 192, and vacuum channel 194,sealing the emulsion well from fluid communication with any otherportion of the droplet generation system (see FIG. 5 for channelpositions). As described previously, this prevents the generateddroplets from passing back into the channel network and thus failing toreach the detection system for a subsequent detection step, and alsofrom potentially contaminating other, reusable portions of the dropletgeneration system. The heat stake also protects droplets and/or carrierfluid from evaporation losses during thermal cycling.

The generated droplets are optionally thermocycled to amplify individualcopies of one or more targets that may be contained within the droplets(e.g., see step 236 of FIG. 9B). Alternatively, droplets may be removedfrom the emulsion well and thermocycled (or otherwise treated) inanother container.

The generated droplets are optionally removed from the emulsion well andtransported to a detection region, where a detection system may beconfigured to detect photoluminescence (such as fluorescence) as thesignature of the presence of one or more particular targets in thedroplets. As mentioned previously, in some cases, the droplets may bethermocycled and/or transported to the detection system while stilldisposed in the emulsion well of the droplet generation system, whereasin other cases the droplets may be extracted from the emulsion wellbefore thermocycling, or after thermocycling and before the detectionstep.

Many variations of microfluidic device 152 generally described above anddepicted in FIGS. 3-9 are possible and fall within the scope of thepresent disclosure. For example, the well component may include anydesired number of wells, such as 32, 96, or 384 wells, among others,allowing 8, 24, or 96 samples, respectively, to be processed intodroplet form with a single system. Similarly, in some cases the channelcomponent may include any desired number of ports and channel networks,allowing multiple samples to be handled with a single channel component.Furthermore, the precise dimensions and shapes of the apertures, wells,channel network, and other elements of the droplet generation system maybe chosen for convenience and performance, and may be different fordifferent applications of the system.

Example 2. Droplet Generation Device with Fluid Pickups and a CarrierManifold

This example describes an exemplary embodiment 252 of microfluidicdevice 52 for fluid processing system 50 (see FIGS. 1, 2, 2A, and 2B).Device 252 is configured for generating an array of emulsions using afixed, dedicated pickup tube for the sample of each emulsion. The devicealso has a carrier manifold that receives carrier fluid for all of theemulsions from an off-device (external) carrier reservoir that isfluidically connected to device 252; see FIGS. 10-18.

FIGS. 10 and 11 show respective assembled and exploded views of device252. The device may have any combination of the features described abovein Section I and/or Example 1. For example, as described above fordevice 152, device 252 includes a sample-holding portion, namely, a wellcomponent 66 defining a plurality of wells. The device also includes afluid-processing portion, namely, a channel component 72 attached to thewell component and defining a plurality of channels, such asmicrochannels, that are fluidically connected to the wells. Elements ofdevice 252 that functionally and/or structurally correspond to those ofdevice 152 generally are identified using the same reference numbers asfor device 152.

Channel component 72 has a base 76 and a cap 78 overlying the base. Thecap may be attached to the base, and the base attached to well component66, in any suitable order. In other words, channel component 72 may beassembled before or after base 76 is attached to well component 66.Channel component 72 provides an array of fluidically connected emulsionproduction units 154. In the depicted embodiment, the device has atwo-by-eight array of units 154 in an SBS-compatible grid.

Cap 78 defines a plurality of pre-formed apertures 254 through whichsample fluid may be introduced from a top side of device 252 to load thedevice with samples before sample-containing emulsions are formed. Eachaperture 254 is aligned with a different sample well 166 of wellcomponent 66 and forms an opening for a sample port 184 defined by base76 and aligned with the sample well. Apertures 254 may be closed with acover after samples are introduced into the sample wells (e.g., see FIG.9 above for device 152).

Cap 78 also may define one or more assembly openings 256 havingcounterpart assembly openings 258, 260 defined respectively by base 76and well component 66. The assembly openings of the different parts maybe aligned with one another to facilitate alignment of the parts duringassembly when manufactured or by the end user. The openings also oralternatively may allow multiple copies of device 252 to be arrayed in aholder having pegs or other protrusions onto which the openings can beplaced. The holder can position the device copies in an array havingdefined locations and spacings of the copies, and thus of all the samplewells (and emulsion wells) of the copies relative to one another.

FIG. 12 shows a top view of well component 66. The well componentdefines a sample well 166 and an emulsion well 170 under each emulsionproduction unit 154 (also see FIG. 11). Each well may have any suitableshape. For example, in the depicted embodiment, each sample well iselongated horizontally and each emulsion well is conical. The wells areformed in a top surface 262 of well component 66, which may be a planartop surface for attachment in a fluid-tight seal to base 76. Wellcomponent 66 of device 252 has no carrier wells (compare with device 152of FIGS. 3-9).

FIGS. 13-15 show various views of base 76. The base has a channelnetwork 264 formed in a top surface 265 of the base. In otherembodiments, the channel network may be formed in a bottom surface ofcap 78 (e.g., see FIGS. 2A and 2B). Channel network 264 forms emulsionproduction units 154 as a fluidically interconnected set. (One of theemulsion production units is shown in FIG. 14.) The fluidinterconnection of units 154 of device 254 is in contrast to device 152of Example 1, which provides an array of fluidically isolated channelnetworks 156 (see Example 1). More particularly, the emulsion productionunits of device 252 are each fluidically connected to a carrier manifold266 of channel network 264). In the depicted embodiment, the carriermanifold includes a larger (wider/deeper) supply channel 268 of thechannel network that extends longitudinally on the top surface of base76, between the two rows of emulsion production units 154. The carriermanifold connects along its length to lateral branch channels of thechannel network, namely, a pair of carrier channels 176, 178 of eachemulsion production unit 154 (see FIG. 14). In other embodiments, onlyone carrier channel (or three or more carrier channels) may extend fromthe carrier manifold to each emulsion production unit. In otherembodiments, the carrier manifold may include a branched supply channelconfiguration with branches each connected to a subset of the carrierchannels of the device.

Carrier manifold 266 is connectable to an external source of carrierfluid via at least one carrier port. In the depicted embodiment, theopposite ends of carrier manifold 266 form carrier ports 270 a, 270 b(see FIG. 13). However, the carrier manifold may form any suitablenumber of carrier ports at any suitable positions along the carriermanifold. Furthermore, the device may have any suitable number ofdistinct and/or separate carrier manifolds.

The carrier ports may be used in any suitable manner. In someembodiments, only one of the carrier ports may be connected to a sourceof carrier fluid during performance of a method of forming droplets withdevice 252. The user may be permitted to select which carrier port isutilized for introduction of carrier fluid into device 252. The othercarrier port may be left closed and may provide a region where trappedair can collect without interfering with droplet generation.Alternatively, a first carrier port may be connected to a source ofcarrier fluid and a second carrier port may be open or opened to permitpre-loading of the carrier manifold with carrier fluid. In this case,carrier fluid is received in the carrier manifold via the first carrierport, and air and/or excess carrier fluid may leave the carrier manifoldvia the second carrier port. The second carrier port may be sealed aftera pre-loading operation and before application of vacuum or pressure todrive droplet generation. In some embodiments, two or more carrier portsmay be connected to the same source of carrier fluid, to allow thecarrier fluid to enter a channel network via two or more distinctcarrier ports.

FIG. 16 shows a sectional view of carrier port 270 a fluidicallyconnected to an external source 272 of carrier fluid 116. The port mayinclude a blind hole 274 formed in the top surface of base 76 andcommunicating laterally with supply channel 268 of the carrier manifold.Blind hole 274 may be wider and/or deeper than supply channel 268. Inother embodiments, supply channel 268 may widen or deepen at anysuitable positions along its length to form one or more carrier ports,and/or the supply channel may be accessed at an arbitrary position alongits length to provide a carrier port. Carrier manifold 266, includingthe carrier ports, may be sealed at the top with cap 78. The cap may bepierced at any time over a carrier port with a hollow piercing element276 that is fluidically connected to the external source of carrierfluid, such as via a conduit 277. Piercing element 276 may be connectedto and/or integrally formed with a gasket 278 capable of forming afluid-tight seal with cap 78 around the piercing element. As a result,the external source of carrier fluid is fluidically connected to carriermanifold 266 via carrier port 270 a, allowing carrier fluid to be pushedand/or pulled into the carrier manifold, indicated by flow arrows at279.

FIG. 14 shows the channels and apertures of one of emulsion productionunits 154. The unit has a similar channel structure to that of device152 (e.g., see FIG. 5). A channel junction 182 that functions as a sitefor droplet generation is formed where a sample channel 174 and carrierchannels 176, 178 meet a droplet channel 180. The unit, as in device152, also has a sample port 184 for addition of sample to a sample well,and an emulsion port 188 for removal of emulsion from an emulsion wellafter the emulsion has been collected in the emulsion well, with eachport formed as a through-hole in base 76. However, the unit has nocorresponding carrier port. Instead, carrier fluid is supplied from aconnected, off-device source. Furthermore, the emulsion production unit,as in device 152, has a sample passage 196 that directs sample upwardfrom an underlying sample well to sample channel 174, and a dropletpassage 200 that directs droplets in carrier fluid from droplet channel180 downward to an underlying emulsion well.

FIG. 17 shows a sectional view taken through a sample well 166 and anemulsion well 170 of device 252. (The channels are too small to bevisible in this view.) A droplet tube 162 projects into emulsion well170 from a body 80 of base 76. The droplet tube is very short, tominimize the chance of collected emulsion re-entering the droplet tube.Droplet passage 200 extends upward through tube 162 to the channelnetwork.

FIG. 18 shows a sectional view taken through a sample well 166. Thesample well is asymmetrically shaped, with a deeper end on the left anda shallower end on the right. Sample tube 162 is extends downward to aposition near the bottom of the sample well at the deeper end thereof.Configuring the sample well to be relatively wide and shallow, asdepicted here, allows sample tube 162 to be shorter and fatter. As aresult, the sample tube can be injection molded integrally with the restof base 76.

Sample port 184 and emulsion port 188 each are connected to anassociated port, namely, a vent port 290 and a vacuum port 292,respectively (see FIGS. 14 and 18). Each of ports 290 and 292 is createdby a respective blind hole 294, 296 formed in the top surface of base 76and connected to port 184 or 188 by a respective channel 298 or 300.

Vent port 290 provides a site for venting sample well 166 after the wellhas been loaded and aperture 254 has been sealed with a cover (also seeFIGS. 10 and 11). Cap 78 can be pierced over vent port 290 with apiercing element to open the port and thus vent the sample well beforeemulsion formation begins. Vent port 290 is spaced from sample port 184by channel 298, which reduces the chance of contamination of thepiercing element with sample when the sample well is vented.

Vacuum port 292 is similar in structure to vent port 290 and provides asite for connecting a vacuum source to the underlying emulsion well viaemulsion port 188. The vacuum port is spaced from emulsion port 188 bychannel 300, which reduces the chance of contaminating the vacuum systemwith emulsion when the cap is pierced over vacuum port 292 to connectthe vacuum source to device 252 and/or when vacuum is applied. Channel300 also allows sealing the emulsion well, after the emulsion isgenerated, by closing off channel 300 through channel deformation, suchas via heat staking through application of heat and, optionally,pressure. Channels 180 and 300 may extend close to each other, to enableheat staking them at the same time in a single operation, such as at aheat stake area 302 extending across both channels, thereby completelysealing the emulsion well after emulsion generation and before thermalcycling. Alternatively, channels 180 and 300 may be heat stakedseparately at the same or different times. Heat staking channels 180 and300 at heat stake area 302 is analogous to heat stake 234 describedabove for the last configuration shown in FIG. 9B.

Example 3. Exemplary Instrument to Interface with a Microfluidic Device

This example describes an exemplary instrument 310 configured tofluidically connect to microfluidic device 252 and exemplary methods ofusing the instrument to generate emulsions; see FIGS. 19-21.

FIGS. 19 and 20 show instrument 310 of microfluidic system 50 fordriving and controlling fluid flow within device 252. Instrument 310 maybe configured for use with any of the microfluidic devices disclosedherein. Any or all of the operations performed automatically byinstrument 310 alternatively may be performed manually by a user.

Instrument 310 may include a support 312 to hold and position device 252with respect to at least one processing head 314. Processing head 314 ispositioned or positionable over device 252, in alignment with thedevice, and is configured to interface with the top side of device 252.In the configuration depicted in FIG. 19, the processing head is in apre-processing, ready position over device 252, before fluid processinghas been initiated and with processing head 314 not yet in contact withthe device. The processing head may be operatively connected to a drivemechanism 316 (a “driver”), a carrier fluid reservoir 318 containingcarrier fluid 116, and a vacuum source 320 including a vacuum pump 322(or other source of vacuum or pressure). A frame may provide sites forattachment, organization, and/or support of other structures of theinstrument.

Processing head 314 has a body 326 and a plurality of piercing elementsconnected to the body (see FIGS. 19 and 20). The piercing elements maybe vertically aligned or alignable with various ports of device 252 toallow the piercing elements to pierce the cover over the port. Piercingthe cover opens the port, such as to form a vent or fluidically connectthe port to carrier fluid reservoir 318 or vacuum source 320.

The piercing elements may be categorized as distinct types, namely,carrier piercing element(s) 328 a, vent piercing elements 328 b, andvacuum piercing elements 328 c according to the type of port opened bythe element. One or more of these types may be arrayed to match thearrangement of corresponding ports of device 252, as illustrated bycomparing FIGS. 13 and 14 with FIG. 20. At least one carrier piercingelement 328 a may be aligned or alignable with at least one carrier port270 a or 270 b (port 270 b is aligned in this example). A plurality ofvent piercing elements 328 b may be aligned or alignable with acorresponding plurality of vent ports 290. (All of the vent piercingelements are aligned with all the vent ports in this example). Aplurality of vacuum piercing elements 328 c may be aligned or alignablewith a corresponding plurality of vacuum ports 292. (All of the vacuumpiercing elements are aligned with all of the vacuum ports in thisexample.) Accordingly, processing head 314 and device 252 may be movedvertically relative to one another to pierce two or more types of portsat the same time. In some embodiments, two or more types of ports may bepierced at different times. For example, the vent ports may be piercedat a different time, such as before or after, the vacuum ports and/orthe carrier port(s). As another example, the carrier port(s) may bepierced at a different time than the vacuum port (e.g., to pre-load thecarrier manifold with carrier fluid). Furthermore, the ports of a giventype may be pierced as two or more sets at different times. Ports may bepierced at different times using, for example, a horizontallypositionable processing head with fewer piercing elements, two or moreprocessing heads (e.g., different heads for the vent ports and thevacuum ports), or a separate tool to manually pierce some of the ports(e.g., the vent ports), among others.

Piercing elements 328 a, 328 b, and 328 c may be on the same processinghead 314 or different processing heads 314 of the instrument. In someembodiments, one or more of the sets of piercing elements may bearranged in an array that matches an array defined by all or a subset ofports of the device. For example, piercing elements 328 b may bearranged in an array that matches an arrangement of two or more ventports 290 of the device. In some embodiments, piercing elements 328 bmay be configured to pierce a plurality of vent ports 290 at the sametime, such as each vent port 290 of the device. Piercing elements 328 cmay be arranged in an array that matches an arrangement of two or morevacuum ports 294 of the device. In some embodiments, piercing elements328 c may be configured to pierce a plurality of vacuum ports 294 at thesame time, such as each vacuum port 294 of the device. In someembodiments, piercing elements 328 b and 328 c may be arranged to piercea plurality of vent ports 290 and a plurality of vacuum ports 294 at thesame time. In some embodiments, piercing elements 328 a and 328 c may bearranged to pierce at least one carrier port 270 a or 270 b and aplurality of vacuum ports at the same time.

Each piercing element may be associated with a gasket 330, which may bededicated to the piercing element or shared among two or more piercingelements (see below). Each piercing element may be hollow or solid.

Carrier piercing element(s) 328 a may be connected to carrier fluidsource 318, indicated by an arrow at 332. Accordingly, each carrierpiercing element 328 a may fluidically connect the carrier fluid sourceto carrier manifold 266 when piercing element 328 a enters the carrierport.

Vent piercing element(s) 328 b may create fluid communication with theatmosphere. Accordingly, each vent piercing element 328 b may functionto connect ambient air outside the device with a vent port 290 and itsassociated sample well when piercing element 328 b opens the vent port(e.g., after the sample port has been covered).

Vacuum piercing element(s) 328 c may be connected to vacuum source 320,indicated by an arrow at 334. Accordingly, each piercing element 328 cmay fluidically connect the vacuum pump to a vacuum port (and itsassociated emulsion well) when the piercing element enters the vacuumport. Head 312 may include a vacuum manifold 336 through which thevacuum piercing elements communicate with a vacuum source.

Drive mechanism 316 moves support 312 and head 314 relative to oneanother. In the depicted embodiment, the drive mechanism causes head 314to move while support 312 remains stationary. In other embodiments, thedrive mechanism may cause support 312 to move while head 314 remainsstationary, among others. The drive mechanism may move head 314 in onlyone dimension, namely, along a vertical axis, such as if the head canperform all of its functions in one position on the device.Alternatively, the drive mechanism may move head 314 in two or threedimensions, indicated at 340, which may permit the head to be positionedon the device a plurality of times to perform different functions and/orto perform the same function multiple times for different subsets of theemulsion production units (e.g., to produce emulsions from differentsubsets of the units in sequence).

The instrument also may be configured to deform channels of device 252,such as by heat staking, to block passage of fluid through the channels.For example, the instrument may have a one or more heating elements thatcan be pressed against the top side of device 252 after emulsionformation to seal each emulsion well by blocking a channel 180 and achannel 300 associated with the emulsion well (see FIG. 14).Accordingly, the heating elements may be arranged in an array thatmatches the spacing of the emulsion production units, and moreparticularly, in an array that matches and is alignable with an array ofheat stake areas 302 defined by channel pairs 180 and 300 (see FIG. 14).The heating elements may be present on head 314 or a different head ofthe instrument, or may be provided by a different instrument. In anyevent, the emulsions may be fluidically isolated in the emulsion wellsby channel deformation and then thermally cycled within the wells. Insome embodiments, a laser may be used to deform the channels.

Carrier fluid reservoir 318 may be vented to allow vacuum pump 322 topull carrier fluid into device 252 from the reservoir. Alternatively, orin addition, the carrier fluid reservoir may be connected to an optionalcarrier pump 342 that drives carrier fluid 116 from the reservoir intothe device, optionally with assistance from vacuum source 320. Thecarrier pump generally is not needed unless it is used to pre-loadcarrier fluid into the device prior to application of vacuum with thevacuum source.

Instrument 310 further may include a processor 344 (interchangeablytermed a controller) in communication with any combination of drivemechanism 316, vacuum pump 322 and/or a valve and/or a pressure gaugetherefor, and/or carrier pump 342, among others. The processor maycontrol and coordinate fluid processing within device 252.

FIG. 21 shows another exemplary processing head 314 a for instrument310. Head 314 a differs from head 314 in having gaskets 330 a, 330 bthat are each shared by a plurality of vacuum piercing elements 328 c.In some embodiments, the gasket may be formed integrally with one ormore piercing elements.

Emulsions may be prepared with device 252 as follows. The stepspresented below may be performed in any suitable order and combination,and each may be performed by the user or instrument 310, as described.Figures showing structures involved in particular steps are referencedbelow.

A sample may be loaded into each of sample wells 166 (see FIGS. 10 and11). Replicates of the sample or different samples may be loaded intothe wells. Each sample may be introduced into a sample well through anaperture 254 of cap 78 and a sample port 184 of base 76 and/or body 80(see FIGS. 10, 11, and 14).

Sample ports 184 may be covered by application of at least one cover.One or more covers may be placed over the sample ports, which may (ormay not) be attached to cap 78 and may (or may not) seal each sampleport. Covering each sample port may reduce the chance ofcross-contamination among the sample wells. In some embodiments, thesample ports may be left uncovered, covered loosely, or covered with apre-perforated cover, among others, which may obviate the need forpiercing a vent port for the sample well.

Device 252 may be placed onto support 312 of instrument 310. In someembodiments, vent ports 290 may be pierced (e.g., with a separate tool)before the device is placed onto the support. In some embodiments, thevent ports may be open when supplied to the user (e.g., pierced by themanufacturer or left uncovered when manufactured).

Processing head 314 and device 252 may be moved relative to each otherto pierce a cover (composed of one or more layers) over ports, which mayconnect a source of carrier fluid, such as carrier reservoir 318, to oneor more carrier ports, and/or connect vacuum source 320 to one or morevacuum ports.

Vacuum may be applied to device 252 with vacuum source 320 to driveemulsion formation. The vacuum may, for example, be applied by opening avalve of the vacuum source to connect the vacuum pump and/or a vacuumchamber to each vacuum port of device to 252. The applied vacuum drawscarrier fluid into carrier manifold 266 and into each of carrierchannels 176, 178 of each emulsion production unit 154 (see FIG. 14).The applied vacuum also draws sample fluid from each sample well into asample channel 174. The channel network is designed to provide greaterfluid impedance for sample travel to each channel junction 182 relativeto carrier fluid travel to the channel junction, such that the carrierfluid reaches the channel junction first. This arrangement ensures thatall of the sample in the emulsion is encapsulated by the carrier fluid,if sufficient carrier fluid is supplied to the channel junction. Theresulting emulsion formed by each emulsion production unit 154 iscollected in a respective emulsion well 170 (see FIGS. 11-14). Eachemulsion can be removed from the emulsion well via the emulsion portafter piercing cap 78, or piercing the emulsion port may be unnecessaryif the emulsion port is not covered during emulsion formation.

Further aspects of exemplary instruments to drive and control dropletgeneration and exemplary methods of droplet generation that may besuitable for the fluid processing systems of the present disclosure aredescribed in the patent documents listed above under Cross-References,which are incorporated herein by reference.

Example 4. Selected Embodiments I

This section describes further embodiments of systems and methods fordroplet generation, presented without limitation as a series of numberedparagraphs.

1. A system for producing droplets, comprising: (A) a well componentincluding a sample well, a carrier fluid well, and a emulsion well; and(B) a chip component configured to be attached to the well component andincluding a sample port configured to provide access to the sample wellwhen the chip component is attached to the well component, a carrierfluid port configured to provide access to the carrier fluid well whenthe chip component is attached to the well component, a droplet portconfigured to provide access to the emulsion well when the chipcomponent is attached to the well component, a first aperture leadinginto a first hollow protrusion configured to extend into the sample wellwhen the chip component is attached to the well component, a secondaperture leading into a second hollow protrusion configured to extendinto the carrier fluid well when the chip component is attached to thewell component, a third aperture leading into a third hollow protrusionconfigured to extend into the emulsion well when the chip component isattached to the well component, and a channel network configured toreceive sample-containing fluid from the sample well via the firsthollow protrusion, to receive carrier fluid from the carrier fluid wellvia second hollow protrusion, to generate an emulsion ofsample-containing droplets suspended in carrier fluid, and to transportthe emulsion to the emulsion well via the third hollow protrusion.

2. The system of paragraph 1, further comprising a penetrable coverconfigured to be applied over a top surface of the chip component.

3. The system of paragraph 1, wherein the well component furtherincludes a vacuum well and the chip component further includes a vacuumport configured to provide access to the vacuum well when the chipcomponent is attached to the well component, and a fourth apertureleading into a fourth hollow protrusion configured to extend into thevacuum well when the chip component is attached to the well component,and wherein the channel network is configured to generate the emulsionin response to negative pressure applied at the vacuum port andcommunicated to the channel network via the fourth hollow protrusion.

4. The system of paragraph 3, further comprising a vacuum sourceconfigured to fit within the vacuum port and to apply negative pressureto the channel network.

5. The system of paragraph 3, wherein the channel network includes avacuum channel configured to communicate vacuum pressure from the vacuumwell to the emulsion well.

6. The system of paragraph 1, wherein the chip component includes asubstantially planar substrate, and wherein the channel network issubstantially planar and is disposed within the substrate.

7. The system of paragraph 6, wherein the channel network includes asample vent channel configured to provide a passage for air from thecarrier fluid port to the sample well.

8. The system of any of paragraphs 1 to 7, wherein the well componentand droplet generation component are repeated to form a regular array,with pairs consisting of a well component and a vertically adjacentdroplet generation component capable of producing distinct sets ofdroplets.

9. The system of paragraph 8, wherein the regular array is a microplatefootprint.

10. A method of generating droplets, comprising: (A) processing asample-containing fluid into (or adding a sample-containing fluid to) asample well of a well component; (B) processing a carrier fluid into (oradding a carrier fluid to) a carrier fluid well of the well component;(C) applying a sealing member over a top surface of a droplet generationchip component attached to the well component; (D) piercing the sealingmember to expose a carrier fluid port formed in the chip component andproviding access to the carrier fluid well; (E) piercing the sealingmember to expose a vacuum port formed in the chip component; (F)inserting a vacuum source into the vacuum port; (G) applying negativepressure to the vacuum port with the vacuum source and thus causingsample-containing fluid to pass from the sample well into a channelnetwork of the chip component via a first hollow protrusion extendingfrom the chip component into the sample well, and causing carrier fluidto pass from the carrier fluid well into the channel network via asecond hollow protrusion extending from the chip component into thecarrier fluid well; (H) generating droplets of sample-containing fluidsuspended in carrier fluid in a droplet generation region of the chipcomponent; and (I) processing the droplets to a emulsion well of thewell component.

11. The system of paragraph 10, wherein the droplets are transportedfrom the chip component to the emulsion well via a third hollowprotrusion extending from the chip component into the emulsion well.

12. The system of paragraph 11, wherein the vacuum port provides accessto a vacuum well of the well component, and wherein negative pressureapplied to the vacuum port is communicated to the chip component via afourth hollow protrusion extending from the chip component into thevacuum well.

13. The system of paragraph 10, further comprising fluidically isolatingthe emulsion well from the other wells of the well component, afterprocessing the droplets to the emulsion well.

14. The system of paragraph 13, wherein fluidically isolating theemulsion well includes applying a heat stake to a portion of the channelnetwork.

15. The system of paragraph 13, further comprising thermocycling thedroplets to cause amplification of target molecules present in thedroplets, and detecting fluorescence radiation emitted by the amplifiedtarget molecules.

16. The system of paragraph 10, wherein piercing the sealing member toexpose the vacuum port is performed by the vacuum source.

17. The system of paragraph 10, wherein the step of adding a sample to asample well is performed after the step of applying a sealing member.

18. The system of paragraph 17, wherein the step of adding a sampleincludes piercing the sealing member to obtain access to the samplewell.

19. A method of generating droplets, comprising: (A) selecting a systemcomprising a droplet generation component, for generating droplets, anda well component, for holding a sample-containing fluid in a samplewell, a carrier fluid in a carrier well, and droplets in a emulsionwell, wherein the droplet generation component is positioned above thewell component, and wherein there is sample-containing fluid in thesample well and carrier fluid in the carrier well; (B) drawingsample-containing fluid and carrier fluid up, against gravity, from thesample well and the carrier well, respectively, to the dropletgeneration component; (C) producing droplets from the sample-containingfluid and carrier fluid with the droplet generation component; and (D)depositing the droplets down, in the direction of gravity, into theemulsion well.

20. The method of paragraph 20, wherein the sample-containing fluid andthe carrier fluid are drawn up to the droplet generation component byrespective input tubes, optionally due to the application of vacuum.

21. The method of paragraph 19 or 20, wherein the droplets are depositeddown into the emulsion well through an output tube.

22. The method of paragraph 21, wherein the droplet tube is disposedabove the emulsion well and dimensioned such that the droplet tube doesnot contact the droplets once they have been deposited in the emulsionwell.

23. The method of any of paragraphs 19 to 22, wherein the input tubesfor the sample-containing fluid and the carrier fluid are disposed abovethe sample-containing fluid and the carrier fluid, and dimensioned tomaintain contact with the sample-containing fluid and carrier fluidduring production of droplets.

24. The method of any of paragraphs 19 to 23, wherein the droplets dripor fall into the emulsion well.

25. The method of any of paragraphs 19 to 24, wherein the systemcomprises the system of any of paragraphs 1 to 9.

Example 5. Selected Embodiments II

This section describes further embodiments of systems and method forfluid processing and/or droplet generation, presented without limitationas a series of numbered paragraphs.

1. A system for fluid processing, comprising: (A) a well; and (B) achannel component including (i) a body including a bottom surfaceattached to the well and a top surface having a microchannel formedtherein, (ii) an input tube projecting into the well from the bottomsurface of the body, and (iii) a passage extending through the inputtube and the body, wherein the system is configured to receive asample-containing fluid in the well such that the sample-containingfluid is in contact with a bottom end of the passage and is retained,with assistance from gravity, below a top end of the passage and out ofcontact with the microchannel until a pressure differential is createdthat drives at least a portion of the sample-containing fluid from thewell via the passage and through the microchannel.

2. The system of paragraph 1, wherein the well is an input well, furthercomprising an output well disposed under the channel component, andwherein system is configured such that the pressure differential drivesat least a portion of the sample-containing fluid from the input well tothe output well.

3. The system of paragraph 2, wherein the input well and the output wellare formed integrally with one another and separately from the body andthe input tube.

4. The system of paragraph 2 or paragraph 3, wherein the body and theinput tube are molded as a single piece, and wherein the input well andthe output well are molded as another single piece.

5. The system of any of paragraphs 2 to 4, wherein the channel componenthas a channel network formed in the top surface of the body, and whereinthe channel network is configured to receive at least a portion of thesample-containing fluid and to generate sample-containing droplets forcollection in the output well.

6. The system of paragraph 5, further comprising a plurality of inputwells and a plurality of output wells, wherein the channel component isconfigured to form a plurality of emulsions from at least onesample-containing fluid disposed in the plurality of input wells and todirect the plurality of emulsions to the plurality of output wells.

7. The system of paragraph 6, wherein each emulsion includes a samecarrier fluid that forms a continuous phase of the emulsion, and whereinthe channel network includes a manifold that supplies the same carrierfluid for each emulsion.

8. The system of any of paragraphs 1 to 7, wherein the channel componentincludes a carrier port connected to the channel network and configuredto receive a carrier fluid that enters the carrier port from above thechannel component in response to the pressure differential.

9. The system of any of paragraphs 1 to 8, wherein the channel componentincludes a cover disposed on the top surface of the body, furthercomprising an instrument configured to pierce the cover and apply avacuum or pressure to the channel component through the pierced cover todrive flow of the sample-containing fluid from the well and through themicrochannel.

10. The system of any of paragraphs 1 to 9, wherein the body and theinput tube are formed integrally with one another and separately fromthe well.

11. The system of any of paragraphs 1 to 10, wherein the channelcomponent defines a sample port over the well and separate from thepassage for introduction of the sample-containing fluid into the well.

12. The system of any of paragraphs 1 to 11, wherein the channelcomponent includes a cover attached in a fluid-tight seal to the topsurface of the body and providing a top wall for the microchannel.

13. The system of paragraph 12, wherein the channel component includesat least one port covered by the cover and configured to be accessed bypiercing the cover.

14. The system of any of paragraphs 1 to 13, wherein the channelcomponent includes a base including the body and the input tube and alsoincludes a cover disposed on the base and at least partially coveringeach of a plurality of ports defined by the base and each fluidicallyconnected to the microchannel.

15. A method of processing fluid, the method comprising: (A) dispensinga sample-containing fluid into a well through a sample port of a channelcomponent including (i) a body having a bottom surface attached to thewell and a top surface with a microchannel formed therein, (ii) an inputtube projecting into the well from the bottom surface of the body, and(iii) a passage extending through the input tube and the body, whereinthe dispensed sample-containing fluid is in contact with a bottom end ofthe passage and is retained, with assistance from gravity, below a topend of the passage and out of contact with the microchannel; and (B)creating a pressure differential that drives at least a portion of thesample-containing fluid from the well via the passage and through themicrochannel.

16. The method of paragraph 15, wherein the step of creating a pressuredifferential causes at least a portion of the sample-containing fluid totravel downward through the body for collection by another well underthe body.

17. The method of paragraph 15 or paragraph 16, wherein the body has achannel network formed in the top surface, and wherein the step ofcreating a pressure differential causes an emulsion of sample-containingdroplets to be generated in the channel network.

18. The method of any of paragraphs 15 to 17, wherein the step ofcreating a pressure differential causes at least a portion of theemulsion to be collected in another well under the body.

19. The method of paragraph 17 or paragraph 18, wherein the step ofdispensing a sample-containing fluid includes a step of dispensing atleast one sample-containing fluid into each of a plurality of inputwells disposed under the body, and wherein the step of creating apressure differential causes a plurality of emulsions to be collected ina plurality of output wells disposed under the body.

20. The method of any of paragraphs 15 to 19, wherein the channelcomponent includes a cover disposed on the top surface of the body andforming a top wall of the microchannel, and wherein the step of creatinga pressure differential includes a step of applying a vacuum or pressureto the channel component at an opening defined by the cover.

21. The method of paragraph 19 or paragraph 20, wherein each of theplurality of emulsions is formed in a different region of the samechannel network.

22. The method of any of paragraphs 19 to 21, wherein droplets of eachemulsion are disposed in a carrier fluid that forms a continuous phaseof the emulsion, and wherein at least a portion of the carrier fluid issupplied to the channel network from a carrier port of the channelcomponent that receives the carrier fluid from a position over thecarrier port in response to the pressure differential.

23. The method of any of paragraphs 15 to 22, wherein the step ofcreating a pressure differential includes a step of piercing a coverdisposed on the top surface of the body to create an opening in thecover, and a step of applying a vacuum or pressure to the channelcomponent at the opening.

24. The method of any of paragraphs 15 to 23, wherein the step ofcreating a pressure differential causes at least a portion of thesample-containing fluid to be collected in an output well, furthercomprising a step of deforming one or more channels that communicatewith the output well to block fluid flow through the one or morechannels and fluidically isolate the at least a portion ofsample-containing fluid collected in the output well, and, optionally, astep of thermocycling the at least a portion of sample-containing fluidcollected in the output well.

25. A microfluidic system for fluid processing, comprising: (A) a wellcomponent including a well; and (B) a channel component including (i) abody having a bottom surface attached to the well component and a topsurface having a microchannel formed therein, (ii) an input tubeprojecting from the bottom surface of the body into the well, and (iii)a port, wherein the body and the input tube collectively define apassage extending upward from an open end of the input tube, through theinput tube and the body, to the top surface, and wherein the system isconfigured to receive a sample-containing fluid in the well via the portfrom above the channel component and to retain the sample-containingfluid below a top end of the passage, with assistance from gravity,until a pressure differential is created that drives at least a portionof the sample-containing fluid out of the well via the passage and intothe microchannel.

26. The system of paragraph 25, wherein the well is an input well,further comprising an output well disposed under the channel component,and wherein system is configured such that the pressure differentialdrives at least a portion of the sample-containing fluid from the inputwell to the output well.

27. The system of paragraph 26, wherein the passage is an input passage,wherein the channel component includes an output tube attached to thebody and projecting from the bottom surface thereof into the outputwell, and wherein the body and the output tube collectively define anoutput passage through which the sample-containing fluid travels forcollection in the output well.

28. The system of any of paragraphs 25 to 27, wherein the well is aninput well, wherein the well component includes an output well, whereinthe body has a channel network formed in the top surface, and whereinthe channel network includes the microchannel and is configured toreceive at least a portion of the sample-containing fluid from the inputwell via the passage and to generate an emulsion of sample-containingdroplets disposed in a carrier fluid for collection in the output well.

29. The system of any of paragraphs 25 to 28, wherein the well componentincludes a carrier well to supply a carrier fluid to the channelnetwork.

30. The system of any of paragraphs 25 to 29, wherein the channelcomponent includes a carrier port configured to receive a carrier fluidfor the channel network from above the channel component, such that thecarrier fluid is introduced into the channel network from the carrierport without contacting the well component.

31. The system of any of paragraphs 25 to 30, wherein the body has achannel network formed in the top surface, and wherein the channelnetwork is configured to receive at least a portion of thesample-containing fluid from the well via the passage and to generatesample-containing droplets disposed in a carrier fluid.

32. The system of any of paragraphs 25 to 31, wherein the well componentincludes a plurality of sample wells to hold a plurality of samples, andwherein the channel network includes a manifold to supply carrier fluidfor generating sample-containing droplets from each of the plurality ofsamples.

33. The system of any of paragraphs 25 to 32, wherein the channelcomponent includes a vacuum port configured to be connected to a vacuumsource.

34. The system of any of paragraphs 25 to 33, further comprising a coverattached in a fluid-tight seal to the top surface of the body andforming a top wall of the microchannel.

35. The system of paragraph 34, wherein the body defines a vacuum portthat is covered by the cover and configured to be connected to a vacuumsource at least in part by piercing the cover.

36. The system of any of paragraphs 25 to 35, further comprising adevice including the well component and the channel component, and evenfurther comprising an instrument including the vacuum source andconfigured to pierce the cover and apply a vacuum to the device suchthat a plurality of emulsions are formed in the device and collected indifferent wells of the well component.

37. The system of paragraph 36, wherein the plurality of emulsions areformed in a same channel network of the device, and wherein theinstrument is configured to supply a carrier fluid to the channelnetwork such that the carrier fluid in introduced into the channelnetwork before contacting the well component.

38. The system of any of paragraphs 25 to 37, wherein the body defines acarrier port that is configured to receive a carrier fluid through anopening in the cover.

39. The system of any of paragraphs 25 to 38, wherein the input tube andthe body of the channel component are formed integrally with oneanother.

40. A system for fluid processing, comprising: (A) a well; and (B) achannel component including (i) a body including a bottom surfaceattached to the well, (ii) an input tube projecting into the well fromthe bottom surface of the body, (iii) a passage extending through theinput tube and the body, and (iv) a microchannel, wherein the system isconfigured to receive a sample-containing fluid in the well such thatthe sample-containing fluid is in contact with a bottom end of thepassage and is retained, with assistance from gravity, below a top endof the passage and out of contact with the microchannel until a pressuredifferential is created that drives at least a portion of thesample-containing fluid from the well via the passage and through themicrochannel.

41. The system of paragraph 40, wherein the body defines a plane, andwherein the microchannel is parallel to the plane.

42. The system of paragraph 40 or paragraph 41, wherein the microchannelis formed in a top surface of the body.

43. The system of paragraph 40 or paragraph 41, wherein the channelcomponent includes a cap attached to a top surface of the body, andwherein the microchannel is formed in a bottom surface of the cap.

44. The system of paragraph 43, wherein the cap is formed by a singlesheet of material.

45. The system of paragraph 43, wherein the cap includes an upper sheetforming a top wall of the microchannel and a lower sheet forming lateralside walls of the microchannel, and wherein the body forms a bottom wallof the microchannel.

46. The system of any of paragraphs 40 to 45, wherein the well is aninput well, further comprising an output well disposed under the channelcomponent, and wherein system is configured such that the pressuredifferential drives at least a portion of the sample-containing fluidfrom the input well to the output well.

47. The system of paragraph 46, wherein the input well and the outputwell are formed integrally with one another and separately from the bodyand the input tube.

48. The system of paragraph 46 or paragraph 47, wherein the body and theinput tube are molded as a single piece, and wherein the input well andthe output well are molded as another single piece.

49. The system of any of paragraphs 46 to 48, wherein the channelcomponent includes a channel network, and wherein the channel network isconfigured to receive at least a portion of the sample-containing fluidand to generate sample-containing droplets for collection in the outputwell.

50. The system of paragraph 49, wherein the channel network is formed ina top surface of the body.

51. The system of paragraph 49 or paragraph 50, further comprising aplurality of input wells and a plurality of output wells, wherein thechannel component is configured to form a plurality of emulsions from atleast one sample-containing fluid disposed in the plurality of inputwells and to direct the plurality of emulsions to the plurality ofoutput wells.

52. The system of paragraph 51, wherein each emulsion includes a samecarrier fluid that forms a continuous phase of the emulsion, and whereinthe channel network includes a manifold that supplies the same carrierfluid for each emulsion.

53. The system of any of paragraphs 46 to 52, further comprising a stepof deforming a region of at least one channel of the channel componentthat provides communication between the input well and the output wellsuch that fluid cannot pass through the least one channel.

54. The system of paragraph 53, wherein the step of deforming includes astep of applying pressure to the channel component over the region ofthe at least one channel.

55. The system of paragraph 53 or paragraph 54, when the step ofdeforming includes a step of applying heat to the channel component overthe region of the at least one channel.

56. The system of any of paragraphs 53 to 55, when the step of deformingincludes a step of melting the channel component at the region of the atleast one channel.

57. The system of any of paragraphs 53 to 56, wherein the step ofdeforming includes a step of creating a longitudinal region of the atleast one channel at which the channel is collapsed.

58. The system of any of paragraphs 53 to 57, when the step of deformingincludes a step of deforming a region of each of two or more channels atthe same time such that fluid cannot pass through any of the two or morechannels.

59. The system of paragraph 58, wherein each of the two or more channelsextend from a same output well.

60. The system of paragraph 59, wherein the step of deforming includes astep of deforming a region of each channel that extends from the outputwell such that the output well is fluidically isolated.

61. The system of any of paragraphs 53 to 60, wherein the step ofdeforming includes a step of fluidically isolating a plurality of outputwells in parallel.

62. The system of any of paragraphs 53 to 61, wherein the step ofdeforming includes a step of deforming a separate region of the channelcomponent for each output well.

63. The system of any of paragraphs 53 to 61, further comprising a stepof thermally cycling fluid in the output well after the step ofdeforming.

64. The system of paragraph 63, wherein the step of thermally cyclingfluid causes amplification of nucleic acid in the fluid.

65. The system of paragraph 63 or paragraph 64, wherein the step ofthermally cycling fluid includes a step of thermally cycling a pluralityof separate emulsions contained by output wells of the system.

66. The system of any of paragraphs 40 to 65, wherein the channelcomponent includes a carrier port connected to the channel network andconfigured to receive a carrier fluid that enters the carrier port fromabove the channel component in response to the pressure differential.

66. The system of any of paragraphs 40 to 65, wherein the channelcomponent includes a cover disposed on the top surface of the body,further comprising an instrument configured to pierce the cover andapply a vacuum or pressure to the channel component through the piercedcover to drive flow of the sample-containing fluid from the well andthrough the microchannel.

67. The system of any of paragraphs 40 to 66, wherein the body and theinput tube are formed integrally with one another and separately fromthe well.

68. The system of any of paragraphs 40 to 67, wherein the channelcomponent defines a sample port over the well and separate from thepassage for introduction of the sample-containing fluid into the well.

69. The system of any of paragraphs 40 to 68, wherein the channelcomponent includes a cover attached in a fluid-tight seal to the topsurface of the body and providing a top wall for the microchannel.

70. The system of paragraph 69, wherein the channel component includesat least one port covered by the cover and configured to be accessed bypiercing the cover.

71. The system of any of paragraphs 40 to 70, wherein the channelcomponent includes a base including the body and the input tube and alsoincludes a cover disposed on the base and at least partially coveringeach of a plurality of ports defined by the base and each fluidicallyconnected to the microchannel.

72. A method of processing fluid, the method comprising: (A) dispensinga sample-containing fluid into a well of a well component via a portdefined by a channel component, the channel component including (i) abody having a bottom surface attached to the well component and a topsurface having a microchannel formed therein, and (ii) an input tubeattached to the body and projecting from the bottom surface of the bodyto a lower inside region of the well, the channel component defining apassage that extends from an open bottom end of the input tube to a topsurface of the body, wherein the sample-containing fluid is in contactwith the open bottom end and is retained below a top end of the passagewith assistance from gravity; and (B) creating a pressure differentialthat drives at least a portion of the sample-containing fluid out of thewell via the passage and into the microchannel.

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in applications claiming priority from this or a relatedapplication. Such claims, whether directed to a different invention orto the same invention, and whether broader, narrower, equal, ordifferent in scope to the original claims, also are regarded as includedwithin the subject matter of the inventions of the present disclosure.Further, ordinal indicators, such as first, second, or third, foridentified elements are used to distinguish between the elements, and donot indicate a particular position or order of such elements, unlessotherwise specifically stated.

We claim:
 1. A method of processing fluid, the method comprising:selecting a microfluidic device including a well component located underand bonded to a channel component, the channel component including (a)an input tube projecting downwardly into an input well of the wellcomponent and having an open bottom end positioned in a lower region ofthe input well, (b) a horizontal microchannel in fluid communicationwith the input tube, and (c) a sample port located vertically above theinput well and communicating with the input well separately from theinput tube and the microchannel; dispensing, with a fluid-transferdevice, a sample-containing fluid into the input well of the wellcomponent via the sample port of the channel component; and creating apressure differential using at least one vacuum/pressure source to driveat least a portion of the sample-containing fluid from the input wellvia the input tube and through the microchannel.
 2. The method of claim1, wherein the channel component defines a flow path extending upwardthrough the input tube from the open bottom end thereof, along thehorizontal microchannel, and downward to an outlet that communicateswith an output well of the well component, and wherein creating apressure differential drives at least a portion of the sample-containingfluid along the flow path from the input well to the output well.
 3. Themethod of claim 1, wherein the channel component comprises a channelnetwork including the microchannel, and wherein the channel network isdisposed in fluid communication with a source of carrier fluid that isimmiscible with the sample-containing fluid, the method furthercomprising generating an emulsion in the channel network in response tothe pressure differential, the emulsion including sample-containingdroplets encapsulated by the carrier fluid.
 4. The method of claim 3,wherein the pressure differential drives at least a portion of theemulsion into an output well of the well component.
 5. The method ofclaim 3, further comprising thermocycling the at least a portion of theemulsion in the output well using a thermocycler.
 6. The method of claim4, wherein the well component comprises input wells including the inputwell and output wells including the output well, wherein dispensingincludes dispensing sample-containing fluid into the input wells of thewell component, and wherein creating a pressure differential drivesgeneration of a plurality of emulsions in the channel network and atleast a portion of each emulsion into one of the output wells of thewell component.
 7. The method of claim 1, wherein the channel componentcomprises a body and a cover attached to a top surface of the body,wherein the cover forms a top wall of the microchannel, and wherein thestep of creating a pressure differential includes a step of applying avacuum or positive pressure to the channel component at an openingdefined by the cover, using a vacuum/pressure source of the at least onevacuum/pressure source.